Project Summary Atrial septation is a critical step in separating the systemic and pulmonary circulations in tetrapods and atrial septal defects are among the most common forms of human congenital heart disease (CHD). The objective of this project is to investigate the cellular and molecular mechanisms required for atrial septation and atrial septal progenitor cell specification. The canonical view of atrial septation is based on intracardiac morphogenetic events. However, recent work in our laboratory and others has engendered a novel paradigm for atrial septation, based on contributions from the second heart field (Mommersteeg et al., 2006; Snarr et al., 2007b; Goddeeris et al., 2008). We have identified a subset of cardiac progenitor cells specific for the atrial septum. Hedgehog signaling in the posterior second heart field marks atrial septal progenitors. These findings imply that atrial septum vs. non-septum cell fate is distinguished at the level of progenitor cell specification rather than by positional information acquired subsequently within the developing atrium. The molecular mechanisms by which these newly characterized progenitor cells are specified and generate the atrial septum are currently unknown. Here, we propose an integrative approach using both forward and reverse genetics to build a molecular pathway required for atrial septation and investigate the specification of atrial septal progenitors. Our specific aims are to (1) Analyze the specification, proliferation, and survival of atrial septal progenitors in wild-type and Hedgehog signaling mutant embryos; (2) Analyze atrial septal progenitors and Hh signaling in cac2 mutant mice; and (3) Identify the molecular basis of cac2, a novel gene required for atrial septation. A greater understanding of the molecular basis of atrial septal progenitor cell specification and function will be delivered at the end of the granting period. This work will contribute to an ongoing paradigm shift in our understanding of the ontogeny of cardiac septal defects.